The present invention relates to a recombinant cell line, specifically one that produces adenoviral E1 and DEF-A and/or DEF-B gene products.
Modified adenoviruses have proven convenient vector systems for investigative and therapeutic gene transfer applications, and adenoviral viral systems present several advantages for such uses. The development of adenoviral vectors rests on an understanding of viral genetics and molecular biology. Structurally, all adenoviral virions are nonenveloped capsids with a number of surface proteins. Through interactions of these capsid proteins with the surface of a host cell, the virus is internalized and encased in an clathrin-coated organelle resembling an endocytotic vessel (Pastan et al., Concepts in Viral Pathogenesis, Notkins and Oldstone, eds.
Springer-Verlag, New York. pp. 141-46 (1987)). The acidic condition within the vesicle alters the surface configuration of the virus, resulting in vesicle rupture and release of the virus into the cytoplasm of the cell, where it is partially freed of associated proteins while being transported to the nucleus. Once an adenoviral genome is within the host cell nucleus, its expression proceeds through a highly ordered and well characterized cascade. Groups of adenoviral genes (i.e., translation units) are typically organized into common transcription units (xe2x80x9cregionsxe2x80x9d), each having at least one distinct promoter. The transcript from each region is processed after transcription to generate the multiple mRNA species corresponding to each viral gene. Generally, the separate regions are either xe2x80x9cearlyxe2x80x9d or xe2x80x9clate,xe2x80x9d although some genes are expressed as early and late.
Cellular transcription factors first bind to the upstream enhancer of the first early (E1A) region of the adenoviral genome. The E1A gene products, in turn, regulate the expression of other early promoters, one of which (E2B) drives the expression of the transcription unit including three early genes involved in adenoviral DNA replication (Doefler, pages 1-95, in Adenovirus DNA, the Viral Genome, and Expression, Nojhoff, Boston, (1986)). These three proteins (the precursor terminal protein (pTP), the single-stranded DNA binding protein (ssDBP), and the DNA polymerase (pol)) form a tight unit with at least three cellular proteins to drive priming and elongation of the viral genome (Bodnar et al., J. Virol., 63, 4344-53 (1989); Schnack et al., Genes Devel., 4, 1197-1208 (1990); Pronk et al., Clomosoma, 102, S39-S45 (1992); Kelly et al., pages 271-308, in The Adenoviruses (H. S. Ginsberg, ed.), Plenum Press, New York (1984)).
Once viral DNA replication commences, the activity of the early promoters declines (Sharp et al., pages 173-204, in The Adenoviruses (H. S. Ginsberg, ed.), Plenum Press, New York (1984)), as does the expression of cellular genes, due to the activity of the viral host shut-off early gene products. Conversely, promoters controlling the expression of the late genes become active beginning with the onset of viral DNA replication (Thomas et al., Cell, 22, 523-33 (1980)). Indeed, DNA replication appears necessary for the expression of some late genes. For example, while the major late promoter (MLP) exhibits some activity at early times, only the promoter proximal genes are expressed (Shaw et al., Cell, 22, 905-16 (1980); Winter et al., J. Virol., 65, 5250-59 (1991)). However, the activity of the MLP sharply increases following the onset of viral DNA replication (Shaw et al., supra), resulting in the expression of all the MLP gene products (Doeller et al., supra; Thomas et al., supra; Nevins et al., Nature, 290, 113-18 (1981)). The structure of this promoter has been extensively characterized (see, e.g., Lu et al., J. Virol., 71(1), 102-09 (1997); Lutz et al., J. Virol., 70(3), 1396-1405 (1996); Reach et al., EMBO J., 10(11), 3439-46 (1991); Reach et al., J. Virol., 64(12), 5851-60 (1990); Brunet et al., Mol. Cell. Biol., 7(3), 1091-1100 (1987); Miyamoto et al., EMBO J., 4, 3563-70 (1985)). In particular, the MLP of the Ad5 serotype has three upstream promoter elements, two downstream elements and an initiator element (INR, SEQ ID NO:1) located at the start site. The three upstream elements are an inverted CAAT box (i.e., GTTA) located 76 base pairs upstream of the start site, an upstream promoter element (UPE, SEQ ID NO:2) located 63 base pairs upstream of the start site, and the TATA box (sequence: TATAAAA), located 31 base pairs upstream of the start site. The two downstream elements are DE1 (SEQ ID NO:3), located 86 base pairs downstream of the start site, and DE2 (SEQ ID NO:4), located 101 base pairs downstream of the start site. These various promoter elements interact with viral and cellular proteins to drive late transcription of the MTLU. For example, two proteins (DEF-A and DEF-B) bind to the downstream elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (Tribouley et al., J. Virol, 68, 4450-57 (1994)), which has been cloned (van Beveren et al., Gene, 16, 179-89 (1981)). In addition, as mentioned, E1A gene products drive some MLP activity during the early stage of infection
Post-transcriptional processing of the major late transcription unit (MTLU) gives rise to five families of late mRNA, designated respectively as L1 to L5 which encode structural components of the viral capsid (Shaw et al., Cell, 22, 905-916 (1980)). These proteins are highly toxic to cells, and they can potentiate immune responses against infected cells (see, e.g., Yang et al., Proc. Nat. Acad. Sci. (USA), 91, 4407-11 (1994)). This immune response leads to tissue swelling and destruction of the transduced cells, shortening the period of time transgenes are expressed in the cells. xe2x80x9cFirst generationxe2x80x9d adenoviral vectors have been engineered to silence the adenoviral genome with the aim of reducing these deleterious effects. Because, as mentioned, the E1A gene products begin the cascade of viral gene expression, the earliest adenoviral vectors lacked functional E1A regions. For example, insertion of an exogenous gene into the E1 region results in recombinant vectors that can express the exogenous gene but not the E1A gene. The recombinant adenoviruses must be propagated either in complementary cells or in the presence of a helper virus to supply the impaired or absent essential E1 products (Davidson et al., J. Virol., 61, 1226-39 (1987); Mansour et al., Mol. Cell Biol., 6, 2684-94 (1986)).
While such first generation viruses have proven effective in several gene transfer applications, they are not optimal for all uses. In particular, because they must be grown in the presence of E1 complementing DNA, at some frequency recombination events can generate a replication competent adenovirus (RCA). RCA contamination of viral stocks is problematic because RCAs can outgrow recombinant stocks and transform host cells. Moreover, at higher multiplicity of infections (m.o.i.s), several adenoviral promoters are active even in the absence of the E1A gene products, which can lead to the production of cytotoxic adenoviral proteins (Nevins, Cell, 26, 213-20 (1981); Nevins et al., Curr. Top. Microbiol. Immunol., 113, 15-19 (1984)). An additional disadvantage of first generation vectors is largely attributable to this background expression of late gene products. For example, such residual late gene expression can potentiate host immune responses eliminating virally transduced cells (see, e.g., Yang et al., supra; Gilgenkrantz et al., Hum. Gene. Ther., 6, 1265-74(1995); Yang et al., J. Virol., 69, 2008-15(1995); Yang et al., J. Virol., 70, 7209-12 (1996)).
One approach for blocking late gene expression is to selectively block viral replication by mutating the virus such that it fails to express one or more of the three E2B enzymes involved in viral DNA replication. However, while E1A-deficient viruses lacking E2B function can be generated, the approach requires the use of complementing cell lines or helper viruses to supply the missing essential gene product (Almafitano, J. Virol., 72(2), 926-33 (1998)). As discussed above, a major drawback to such an approach is that recombination events within packaging cells between such vectors and complementing genes can generate RCAs. Moreover, of the three E2B genes, it is not currently possible to propagate a virus lacking the ssDBP gene entirely because the required co-expression of the complementing ssDBP and E1A gene products in the same packaging cell is lethal (Klessig et al., Mol. Cell Biol., 4, 1354-62 (1984)). A vector has been constructed that has a temperature sensitive mutation in the ssDBP gene (Engelhardt et al., Proc. Nat. Acad. Sci. (USA), 91, 6196-6200 (1994)). In some systems, this vector has resulted in longer gene expression and reduced immune-inflammatory response than first generation vectors (Yang et al., Proc. Nat. Acad. Sci. (USA), 92, 7257-61 (1995); Engelhardt et al., Hum. Gene Ther., 5, 1217-29 (1994)). However, the temperature-sensitive mutation is imperfect, permitting some basal ssDBP activity at core body temperature, especially at high m.o.i.s (Yang et al., Proc. Nat. Acad. Sci. (USA), 92, 7257-61 (1995)). In addition to these drawbacks, the approach of disrupting the E2B region also impacts the MTLU because the three E2B genes lie on the chromosomal strand directly opposite the L1-L5 genes and the MLP, (see, e.g., Almafitano et al., Gene Ther., 4, 258-63 (1997)).
In view of the foregoing problems, there exists a need for a recombinant adenovirus, specifically a virus exhibiting reduced propensity to generate RCAs within packaging cells and less able than first generation vectors to express late viral gene products in a host cell.
The present invention addresses the aforementioned need by providing a recombinant adenovirus which, aside from lacking E1 gene expression, has a mutated MLP. The mutation in the MLP greatly attenuates L1-L5 gene expression in nonpermissive host cells. Thus, such recombinant adenoviruses are less able than first generation vectors to express late viral gene products in a host cell. Moreover, many such recombinant adenoviruses can be grown in packaging cells without the presence of DNA complementary to the wild-type adenoviral MLP, thus substantially reducing the probability for generating RCAs.
The present inventive recombinant adenoviruses will prove highly useful in biological research. Specifically, the invention provides reagents and methods enabling biologists to more easily study viral molecular genetics and cytotoxicity. Additionally, the present invention provides reagents and methods pennitting biologists to investigate the cell biology of viral growth and infection. Furthermore, the recombinant adenoviruses of the present invention will equip the biologist with novel tools for investigating molecular and cellular biology of gene expression and regulation in novel genetic backgrounds. Such studies, for example, can focus on the interaction between gene products in a defined or selected cellular background, the ability of transcription factors to transregulate gene expression via promoter, repressor, or enhancer elements engineered into the adenovirus, etc. The present inventive recombinant adenoviruses also will prove highly useful as gene transfer vehicles for research or in the clinical setting. Specifically, the adenovirses of the present invention are useful vectors for introducing transgenes into tissue culture cells or into the cells of animals to study development or repair of tissues. The vectors will find application as well in treating diseases through the transfer of therapeutic genes.
These and other advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.